An immunoassay is a method of measuring the amount of target substances making use of the affinity between an antigen and an antibody, namely an antigen-antibody reaction. The antigen-antibody reaction exhibits the highest discriminably of target substances and has the most variety among biological phenomena known conventionally. For this reason, much attention is drawn to the immunoassay that enables direct measurement of target substances from a biological sample including a large variety of biomolecules without isolating and purifying the target substances.
FIG. 1 is a flow diagram for explaining one example of immunoassays. First, a sample solution 5 containing target substances 4 is added into a chamber 1 to which antibodies 2 are fixed (A1). Since each antibody 2 has an antigen-binding site for the target substances 4, the addition causes antigen-antibody reactions between the target substances 4 and the antibodies 2. Next, the chamber 1 is washed using a solution such as a buffer solution (A2). Impurities 3 possibly contained in the sample solution are removed from the chamber 1. Second antibodies 7 are then added into the chamber 1 (A3). Each second antibody 7 has an antigen-biding site that is not identical to the site of each antibody 2. The addition of the second antibodies 2 causes antigen-antibody reactions between the target substances 4 bound to the antibodies 2 and the second antibodies 7. Each second antibody 7 is labeled with a known labeling substance 6 such as a fluorescent substance, a radioactive substance and an enzyme. The chamber 1 is then washed again using a solution such as a buffer solution (A4) for removing second antibodies 7 that are not bound to the target substances 4 from the chamber 1. The amount of the target substances 4 is then calculated by measuring the amount of complexes, each composed of the antibody 2, the target substance 4 and the second antibody 7, remained in the chamber 1, more specifically, the amount of the labeling substances 6 labeling the second antibodies 7 of the complexes (A5).
FIG. 2 is a flow diagram for explaining another example of immunoassays. In this example, a solution containing labeled target substances 4b at a predetermined concentration is added with a sample solution containing target substances 4a into the chamber 1 (B1). The labeled target substances are mimic targets and each labeled target substance has an epitope identical to epitopes of the target substances 4a and is labeled with the labeling substance 6. The addition causes competitive antigen-antibody reactions are progressed in the chamber 1 between the antibodies 2 and the target substances 4a and between the antibodies 2 and the labeled target substances. The chamber 1 is then washed using a solution such as a buffer solution (B2) for removing substances such as impurities 3 possibly contained in the sample solution and unreacted labeled target substances from the chamber 1. The amount of the target substances 4a is then calculated by measuring the amount of the labeled target substances added into the chamber and the amount of complexes, composed of the antibody 2 and the labeled target substance, remained in the chamber 1, more specifically, the amount of the labeling substances 6 labeling the labeled target substances of the complexes (B3).
The immunoassay is not limited to the two examples mentioned above, and also can be performed by other assaying methods. The amount of the target substances in the sample solution is calculated on the basis of the amount of the labeling substances that reflects the amount of the target substances in any assaying method. Examples of the method of measuring the amount of the labeling substances include a method using a means of measuring the amount optically. This method requires a light source and a fluorescence detector as described in the following paper: Tadayuki Tsukatani and Kiyoshi Matsumoto, “Quantification of L-Tartrate in Wine by Stopped-Flow Injection Analysis Using Immobilized D-Malete Dehydrogenase and Fluorescence Detection”, Analytical Sciences, March 2000, vol. 16, pp. 265-268, and a device for the optical measurement is not easy to downsized and downscaled.
Much attention is drawn to a method employing an electrochemical means from the viewpoint of downsizing and downscaling a measurement device employed in the immunoassays as well as performing the assays in safety, easily and with high accuracy. JP 2(1990)-62952A and JP 9(1997)-297121A, for example, disclose a biosensor for measuring the amount of target substances in a sample making use of an enzymatic cycling reaction system that employs alkaline phosphatase as a labeling substance and potassium hexacyanoferrate(III) (potassium ferricyanide) as an electron mediator.
FIG. 3 is a diagram for explaining the enzymatic cycling reaction system employed in biosensors of JP 2(1990)-62952A and JP 9(1997)-297121A. This enzymatic cycling reaction system is composed of first to third reactions induced in a reaction solution containing alkaline phosphatase, oxidized nicotinamide adenine dinucleotide phosphate (NADP), ethanol, alcohol dehydrogenase, diaphorase, and potassium ferricyanide that is to be a substrate of diaphorase. In the first reaction, NADP is dephosphorylated by alkaline phosphatase and then converted into oxidized nicotinamide adenine dinucleotide (NAD). In the second reaction, a redox reaction through catalysis of alcohol dehydrogenase reduces the first reaction induced-NAD into reduced nicotinamide adenine dinucleotide (NADH) and oxidizes ethanol into acetaldehyde. In the third reaction, the second reaction-induced NADH is oxidized by potassium ferricyanide through catalysis of diaphorase and then converted into NAD, and the potassium ferricyanide is converted into potassium hexacyanoferrate(II) (potassium ferrocyanide). NADP may be replaced with reduced nicotinamide adenine dinucleotide phosphate (NADPH). Voltage application to the reaction solution converts the potassium ferrocyanide into potassium ferricyanide. Since the first to the third reactions are progressed in the reaction solution, the amount of the potassium ferrocyanide generated by the third reaction reflects the amount of the alkaline phosphatase contained in the reaction solution. The amount of the alkaline phosphatase is thus measured through a measurement of the amount of an oxidation current generated by the conversion from the potassium ferrocyanide into the potassium ferricyanide.
Long-term retainment of reagents involved in enzymatic cycling reactions in a chip is significant to provide a biosensor chip. The enzymatic cycling reaction system using alcohol dehydrogenase requires ethanol as mentioned above. Ethanol is not easily retained in the chip for a long time due to the high volatility. For this reason, a biosensor chip is not realized easily with the enzymatic cycling reaction system employing alcohol dehydrogenase.